Team:HokkaidoU Japan/Notebook/September14

From 2010.igem.org

(Difference between revisions)
(→Team:HokkaidoU_Japan/ProtocolsEthanol precipitation)
 
Line 88: Line 88:
→Incubated at 37C for 120 min<br>
→Incubated at 37C for 120 min<br>
→Added Sample Buffer 10 uL and electrophoresed
→Added Sample Buffer 10 uL and electrophoresed
-
* pSB1C3band wasn't visible
+
* pSB1C3 band wasn't visible
→ Other 2 were gel extracted
→ Other 2 were gel extracted
* Diluted that final volume would be 40 uL
* Diluted that final volume would be 40 uL
Line 95: Line 95:
[[Image:HokkaidoU Japan 20100914b.jpg‎|200px|right|thumb|Electrophoresis after ethanol precipitation]]
[[Image:HokkaidoU Japan 20100914b.jpg‎|200px|right|thumb|Electrophoresis after ethanol precipitation]]
-
* Added ethanol acetate[3M] 4 uL to 40 uL of previously purified DNA solutuion
+
* Added ethanol acetate[3 M] 4 uL to 40 uL of previously purified DNA solution
-
* Added EtOH[100%] 44 uL friezed in dry ice
+
* Added EtOH[100%] 44 uL froze in dry ice
-
* Melted and centrifuged at 15,996rpm, 4C 5 min
+
* Melted and centrifuged at 15000 rpm, 4C 5 min
* Discarded supernatant and added EtOH[70%] 100 uL rinsed
* Discarded supernatant and added EtOH[70%] 100 uL rinsed
* Centrifuged at 15,996rpm, 4C 5 min
* Centrifuged at 15,996rpm, 4C 5 min
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|-
|-
|style="border-top:1px solid #996;"|'''Total'''
|style="border-top:1px solid #996;"|'''Total'''
-
|style="border-top:1px solid #996;"|'''12.4 uL'
+
|style="border-top:1px solid #996;"|'''12.4 uL'''
|}
|}
*50 uL were transformed by electroporation and remaining 50 uL by heat shock (chemical)
*50 uL were transformed by electroporation and remaining 50 uL by heat shock (chemical)

Latest revision as of 08:20, 27 October 2010

Notebook

Digestion and ligation of pSB1C3, araC Promoter and GFP

Digestion

Electrophoresed digestion product

Used pSB1C3 which was PCRed september 6th. DNA concentration was 2256 ng/uL. So diluted 20 times.

Reagent Amount
10x M buffer 5 uL
DW 34
pSB1C3 1
BSA 5
EcoR I 2
Pst I 3
Total 50 uL
Reagent Amount
10x M buffer 5 uL
DW 27
Promoter 8
BSA 5
EcoR I 3
Spe I 2
Total 50 uL
Reagent Amount
10x M buffer 5 uL
DW 32
GFP 3
BSA 5
Xba I 4
Pst I 1
Total 50 uL

→Incubated at 37C for 120 min
→Added Sample Buffer 10 uL and electrophoresed

  • pSB1C3 band wasn't visible

→ Other 2 were gel extracted

  • Diluted that final volume would be 40 uL

Ethanol precipitation

Electrophoresis after ethanol precipitation
  • Added ethanol acetate[3 M] 4 uL to 40 uL of previously purified DNA solution
  • Added EtOH[100%] 44 uL froze in dry ice
  • Melted and centrifuged at 15000 rpm, 4C 5 min
  • Discarded supernatant and added EtOH[70%] 100 uL rinsed
  • Centrifuged at 15,996rpm, 4C 5 min
  • Discarded supernatant and dried
  • Diluted in 2 uL of TE each
  • Anticipated concentrations

promoter: 125 ng/2uL
GFP: 106 ng/2uL

  • pSB1C3 was cut(EcoR1, Pst1) on September 6th
  • Did ethanol precipitation and electrophoresed to check concentration
  • It was about 20 ng/uL

→Used 2 uL of it

Ligation

Reagent Amount
pSB1C3 2 uL
promoter 2 uL
GFP 2 uL
Mighty Mix 6 uL
T4 ligase 0.4 uL
Total 12.4 uL
  • 50 uL were transformed by electroporation and remaining 50 uL by heat shock (chemical)
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